Pathologic thrombosis is a leading cause of morbidity and mortality in the developed world. While predisposing genetic risk factors have been identified, unknown modifier genes contribute to the variable disease severity and penetrance observed among patients and families with and without thrombophilic risk factors. Understanding of such modifiers could help classify patients at higher risk for thrombosis and recurrence, and might also be informative as risk factors modulating the severity of bleeding disorders. This project will take advantage of powerful genetic tools, including genome editing nucleases, next generation sequencing, and the zebrafish model to conduct a large scale evaluation of hemostasis regulatory genes with the potential to modify the severity of human coagulation disorders. In preliminary studies, we have produced a model of induced and spontaneous thrombosis by targeted mutagenesis of the zebrafish antithrombin III (at3) gene using zinc finger nucleases, and shown that this results in consumptive coagulopathy and spontaneous thrombosis. We have developed a number of additional coagulation factor mutants using robust genome editing nucleases (TALENS and CRISPR/Cas), and conducted a chemical mutagenesis screen that has identified potential suppressor mutant lines harboring prospective thrombosis modifier genes. In Specific Aim 1, we will use CRISPR/Cas to evaluate the effect of reduction of individual coagulation factors on spontaneous thrombosis in the context of at3 deficiency. In Specific Aim 2, we will evaluate complex multigene epistatic interactions of these coagulation factor molecules using CRISPR/Cas at a level and throughput that is not possible in mammalian systems with current technology. Specific Aim 3 will isolate the modifier genes underlying the suppressor mutants generated in our mutagenesis experiment using next generation sequencing technologies. These studies will identify critical interactions amongst known coagulation factors as well as unknown modifiers of these pathways, which will be candidates for enhanced diagnosis and therapy of human thrombotic and hemorrhagic diseases.